Diabetes mellitus is a significant health problem, affecting over 18 million people in the United States alone. Mutations in several hepatic nuclear factors have been linked to early onset non-insulin dependent diabetes mellitus (MODY), underscoring the importance of the hepatic transcription factors for glucose homeostasis. In the previous grant cycle, we have employed tissue-specific gene ablation to demonstrate the essential function of Foxa2 (previously known as HNF3(3) in the integration of the transcriptional response of the hepatocyte to fasting. In addition, Foxa2 has been proposed as a major mediator of insulin signaling in hepatocytes. We propose the following three Aims: In Aim 1, which is the direct result of the interactions within the PO1 with Dr. Birnbaum, we will test the hypothesis that Foxa2 is the major mediator of insulin signaling via AKT2 using genetic means. We will derive mice which are deficient for both AKT2 and Foxa2 in hepatocytes to test if Foxa2 is indeed required to establish the AKT2 mutant phenotype. In Aim 2, we will collaborate with Dr. Ahima to investigate the combined role of Foxa1 AND Foxa2 in hepatic metabolism. This aim is based on our discovery that Foxal and Foxa2 act jointly to enable the hepatogenic program during fetal devlopment. We hypothesize that the two genes also cooperate in transcription in the adult hepatocyte. We will use simulatenous conditional gene ablatation for Foxa1 and Foxa2 combined with physiological and genomics approaches to test our hypothesis that the two genes open chromatin to enable binding of hormone-dependent transcription factors like CREB and GR. In Aim 3, we will address a recent controversy concerning the regulation of hepatic gluconeogenesis by cAMP. Currently, two conflicting models exist regarding the regulation of the transcription factor CREB in hepatocyte. The first proposes that PKA-dependent phosphorylation of CREB is required for recruitment of the co-activator CBP/p300 and subsequent activation of target genes, while the second invokes PKA-dependent translocation of the novel co-activator TORC2 from the cytoplasm to the nucleus as the central regulator of CREB-dependent transcription. We will address the relative importance of both pathways in hepatic glucose homeostasis by genetic means. We will develop mouse models carrying a Ser/Ala mutation in the PKA-phosphorylation site of CREB or a hepatocyte-specific ablation of TORC2, and analyze the consequences to glucose metabolism both in vivo and in isolated hepatocytes. Together, these studies will further our understanding of the transcriptional regulation of hepatic metabolism and in insulin action.